The main objective of the proposed research is to learn how the various components of chromatin interact to regulate gene expression in eukaryotic organisms. The ribosomal RNA genes (rDNA) in the ciliated protozoan Tetrahymena are being used as a model system. This rDNA chromatin is thought to have nucleosome structure similar to that of the bulk chromatin of higher organisms. The in vivo chromatin structure is being probed by photochemical crosslinking of the DNA in living cells with psoralen derivatives. The crosslinking is known to occur mainly in the accessible regions of DNA between nucleosomes. After purification of the rDNA, electron microscopy is used to map the positions of psoralen crosslinks and thereby show the locations of chromosomal proteins on gene and spacer regions. Isolation of the rDNA chromatin in its transcriptionally active state is being attempted. The structure and function of the chromatin in different states of transcriptional activity will be compared to identify the roles of histones, nonhistone proteins, and RNA polymerase in the regulation of rRNA synthesis. A second objective of the research is to further characterize the interaction of psoralen derivatives with DNA and DNA-protein complexes.